We have reproduced the process of protein transport across and of protein integration into the mammalian endoplasmic reticulum membrane by the use of proteoliposomes reconstituted from pure phospholipids and purified membrane proteins. The transport of some proteins requires only two membrane protein complexes: the signal recognition particle receptor, needed for targeting of a nascent chain to the membrane, and a novel complex, the Sec61p complex, that consists of Sec61p and two smaller polypeptides. The translocation of other proteins also needs the presence of the translocating chain-association membrane (TRAM) protein. The integration of two membrane proteins of different topologies into the membrane does not require additional components. These results indicate a surprising simplicity of the basic translocation machinery. They suggest that the Sec61p complex binds the ribosome during translocation and forms the postulated protein-conducting channel.